Stroke is the second leading cause of death worldwide (Woodruff et al., 2011; Moskowitz et al., 2010), and the third leading cause of mortality in Australia (Senes, 2006). In addition to the high rate of mortality, there is an extremely high incidence of morbidity in stroke survivors, making it the leading cause of disability in industrialised countries (Liu et al., 2012).
Within a few minutes of the onset of cerebral ischemia, the infarct core is mortally injured and undergoes necrotic cell death. The infarct core (or striatal region) of the stroke is commonly considered unsalvageable. The necrotic core is surrounded by a zone of less severely affected tissue known as the ischemic penumbra or peri-infarct zone, which is potentially salvageable via post-stroke therapy (Woodruff et al., 2011). The use of recombinant tissue plasminogen activator (rtPA) to help restore blood flow to the ischemic region is, to date, the only approved agent for treatment of acute ischemic stroke. It is used in only 3-4% of all stroke patients (Besancon et al., 2008) due to its narrow therapeutic window and the risk of inducing intracranial haemorrhage (Moskowitz et al., 2010). There is clearly a need for more effective neuroprotective agents. A longer time window for therapeutic intervention would also be advantageous.
During cerebral ischemia, severe oxygen depletion occurs, compelling the brain to switch from oxidative phosphorylation to anaerobic glycolysis, leading to acidosis as a result of increased lactate levels. The extracellular pH can fall from ˜7.3 to 6.0-6.5 in the ischemic core under normoglycemic conditions, and can drop to below 6.0 during severe ischemia (Xiong et al., 2004; Isaev et al., 2008). The drop in extracellular pH activates acid sensing ion channels (ASICs), and activation of these channels is thought to play a critical role in stroke-induced neuronal injury. Numerous studies have demonstrated a direct correlation between brain acidosis and infarct size (Xiong et al., 2007).
ASICs were discovered in the late 1990s, almost 20 years after the observation that sensory neurons depolarise in response to a sudden drop in pH (Krishtal, 2003). Although they belong to the epithelial sodium channel/degenerin family of receptors, they are distinguished by their restriction to chordates, predominantly neuronal distribution, and activation by decreases in extracellular pH (Gründer and Chen, 2010). Alternative splicing of four ASIC-encoding genes leads to the expression of six subunits (ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3 and ASIC4) that combine to form hetero- or homo-trimeric channels that differ in their pH sensitivity, kinetics, and susceptibility to desensitisation (Wemmie et al., 2006).
Postsynaptic ASIC1a channels are the dominant ASIC subtype in mammalian brain (Xiong et al., 2004; Li et al., 2010). The pH for half-maximal activation (pH0.5) of ASIC1a is 6.6 in human cortical neurons (Li et al., 2010) and 6.4 in rat Purkinje neurons, and consequently they are robustly activated by the decrease in extracellular pH that occurs during cerebral ischemia. Importantly, homomeric ASIC1a channels can mediate the uptake of Ca2+ in addition to Na+ and protons (Gründer and Chen, 2010). Thus, brain ASIC1a can contribute to the intracellular Ca2+ overload that occurs during stroke, and the proton permeability of ASIC1a may be at least partly responsible for the precipitous drop in intracellular pH from ˜7 to as low as 6.15 during cerebral ischemia (Isaev et al., 2008).
It is now known that cerebral acidosis activates ASIC1a and that this activation is a major contributor to the neuronal damage resulting from stroke (Xiong et al., 2004; Xiong et al., 2007; Wang et al., 2011; Leng et al., 2013). For example, in rodent models of cerebral ischemia, infarct size and neurological deficits are greatly reduced by knockout or pharmacological blockade of ASIC1a (Xiong et al., 2004).
The most potent and selective blocker of ASIC1a described to date is PcTx1 (also known as π-theraphotoxin-Pc1a), a 40-residue peptide isolated from the venom of the Trinidad Chevron tarantula, Psalmopoeus cambridgei. PcTx1 inhibits rat ASIC1a (rASIC1a) with an IC50 of ˜1 nM but it does not inhibit other ASIC homomers or heteromers. In a rat model of transient focal ischemia (middle cerebral artery occlusion; MCAO), intracerebroventricular (i.c.v.) injection of P. cambridgei crude venom (that is, without isolating the pure ASIC1a inhibitory peptide) reduced infarct size by 60%. Consistent with this being an effect mediated by ASIC1a, infarct size was similarly reduced by 61% in ASIC1−/− mice (Xiong et al., 2004). Also, infarct size was reduced by ˜30% when P. cambridgei crude venom was delivered i.c.v. as late as five hours after MCAO (Pignataro et al., 2007).
Whilst PcTx1 appears to be a promising lead molecule for the development of neuroprotective agents for the treatment of stroke, there remains a significant need for compounds that can form the basis of an effective neuroprotective therapy. Given the delay in clinical presentation following stroke, a longer time window for therapeutic intervention would also be advantageous.
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